Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples
Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples
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DOI：
10.1016/j.mcp.2015.08.006
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发表时间：
2015-12-01
影响因子：
3.3
通讯作者：
Azzi, Alberta
中科院分区：
文献类型：
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作者：
Arvia, Rosaria;Corcioli, Fabiana;Azzi, Alberta
不同的病毒可能导致呼吸道感染的相似临床表现。因此，呼吸道病毒性疾病的病原学诊断需要检测大量病毒。在本研究中，使用 EvaGreen 嵌入染料开发了 6 种双重实时 PCR 检测方法，用于检测 12 种导致呼吸道疾病的主要病毒：甲型和乙型流感病毒、肠道病毒（包括肠道病毒属和鼻病毒属）、呼吸道合胞病毒、人类偏肺病毒、冠状病毒 I 组（其中 CoV 229E 和 CoV NL63 是其中的一部分）和 II 组（包括 Coy OC43 和 CoV） HKU1)、副流感病毒 1、2、3 和 4 型、人类腺病毒和人类博卡病毒。每个双链体反应的 2 个目标病毒可通过其扩增子的解链温度来区分。 6 种双重实时 PCR 检测用于对 157 名患者的 202 份呼吸道样本进行诊断。 157 个样本是咽喉拭子，45 个样本是支气管肺泡灌洗液。通过与商业化、经过验证的检测进行比较，确认了双重 PCR 检测的结果；此外，阳性结果还通过测序得到证实。双链 PCR 检测的分析灵敏度从 10(3) 拷贝/ml 到 10(4) 拷贝/ml 不等。仅副流感病毒 2 的含量为 10(5) 拷贝/毫升。来自 55 名患者（30 名儿童和 25 名成人）的 70 份临床样本 (35%) 对 1 种或多种病毒呈阳性。在成年患者中，甲型流感病毒是最常检测到的呼吸道病毒，其次是鼻病毒。相比之下，呼吸道合胞病毒是儿童中最常见的病毒，其次是肠道病毒、甲型流感病毒和冠状病毒NL63。由于样本/患者数量较少，我们无法得出任何流行病学结论。总而言之，本研究的结果表明本研究中描述的 6 种双重 PCR 检测方法灵敏、特异且经济高效。因此，该测定对于在核酸提取后直接从临床样本中鉴定主要呼吸道病毒特别有用，并且还可以筛选大量患者进行流行病学研究。 (C) 2015 Elsevier Ltd. 保留所有权利。
Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including Coy OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be particularly useful to identify the main respiratory viruses directly from clinical samples, after nucleic acid extraction, and, also, to screen a large number of patients for epidemiological studies. (C) 2015 Elsevier Ltd. All rights reserved.